In previous experiments we showed that the amount of Glut-1 glucose transporter protein and mRNA present in cells does not correlate with the ability of cells to accumulate 2-fluoro-2-deoxyglucose (FDG). We have since further characterized other biochemical properties of A431 cells (which express high levels of Glut-1) and T47D cells (which express low levels of Glut-1). Neither cell line expresses other glucose transporter isoforms at detectable levels by Northern blot analysis.We have now over-expressed the Glut-1 glucose transporter in both these cell lines. In both lines we obtained levels of expression, at the mRNA level, that were 5 to 75 times higher than the wild-type cells. In A431 cells the amount of total Glut-1 protein was unchanged, suggesting some post-translational control of Glut-1 protein levels in these cells. In T47D cells, a 10-fold increase in the amount of total Glut-1 protein did not increase FDG uptake, while greatly increasing the ability of cells to transport 3-O-Methyl glucose, a glucose analogue that is not phosphorylated.Studies of the mitochondrial phosphorylating activity towards FDG for these two cells show that mitochondria of A431 cells, which accumulate FDG at a lower rate than T47D cells, show a lower affinity for FDG (Km ~ 300 'M) compared with mitochondria of T47D cells (Km ~ 100 'M), and that for equal amounts of mitochondrial protein, the maximum velocity (Vmax) of FDG phosphorylation is two times faster for T47D preparations than for A431 preps. These data suggest a tighter control of FDG phosphorylating activity on FDG uptake, rather than FDG transport activity.At the mRNA level, however, T47D cells show lower steady state levels of both the Hexokinase I and II transcripts, suggesting that phosphorylating activity does not correlate with the level of gene expression, or that, putatively, another hexokinase isoform may be present in T47D, thus providing higher levels of FDG phosphorylation in these cells.